Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present-Future

γδ T-cells directly recognize and kill transformed cells independently of HLA-antigen presentation, which makes them a highly promising effector cell compartment for cancer immunotherapy. Novel γδ T-cell-based immunotherapies, primarily focusing on the two major γδ T-cell subtypes that infiltrate tumors (i.e. Vδ1 and Vδ2), are being developed. The Vδ1 T-cell subset is enriched in tissues and contains both effector T-cells as well as regulatory T-cells with tumor-promoting potential. Vδ2 T-cells, in contrast, are enriched in circulation and consist of a large, relatively homogeneous, pro-inflammatory effector T-cell subset. Healthy individuals typically harbor in the order of 50-500 million Vγ9Vδ2 T-cells in the peripheral blood alone (1-10% of the total CD3+ T-cell population), which can rapidly expand upon stimulation. The Vγ9Vδ2 T-cell receptor senses intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of mevalonate pathway dysregulation or upon pharmaceutical intervention. Early clinical studies investigating the therapeutic potential of Vγ9Vδ2 T-cells were based on either ex vivo expansion and adoptive transfer or their systemic activation with aminobisphosphonates or synthetic phosphoantigens, either alone or combined with low dose IL-2. Immune-related adverse events (irAE) were generally \mild, but the clinical efficacy of these approaches provided overall limited benefit. In recent years, critical advances have renewed the excitement for the potential of Vγ9Vδ2 T-cells in cancer immunotherapy. Here, we review γδ T-cell-based therapeutic strategies and discuss the prospects of those currently evaluated in clinical studies in cancer patients as well as future therapies that might arise from current promising pre-clinical results.


INTRODUCTION
In humans, gd T-cells represent 1 to 10% of total CD3 + T-cells (1,2), and express a combination of either of 7 different Vg TCR chains (Vg2, 3, 4, 5, 8, 9, and 11), paired with either of 4 Vd (Vd1, 2, 3, and 5) chains (2)(3)(4). gd T-cells are considered to bridge the innate and adaptive immune systems (3). Activated gd T-cells display strong cytotoxic activity through the release of granzyme B and perforin, by membrane bound TRAIL and Fas (CD95) ligands or production of IFNg or TNFa to amplify the immune response (12), thereby counteracting tumor development. Using gd T-cell-deficient mice in a cutaneous carcinogenesis model, gd T-cells were first shown to prevent malignancy formation (5). High gd T-cell frequency in tumor infiltrates from cancer patients correlates with better clinical outcome in different malignancies (6)(7)(8)(9)(10) and gd T-cells were identified as the prognostically most favorable immune cell subset in tumor infiltrates from 18,000 tumors across 39 malignancies (11). A more recent study confirmed the relative abundance of Vg9Vd2 T-cells in TILs and their association with improved patient outcome (12). These results highlight the relevance of gd T-cells in tumor control and their potential for cancer therapy. gd T-cells express several receptors shared with natural killer (NK) cells that participate in enhanced tumor cell recognition of which FcgRIIIa (CD16a), DNAM-1, and NKG2D are a few examples (13) (Figure 1A). The complete repertoire of antigens recognized by gd-TCRs and the specificity of each gd T subset is still not fully understood.
In this review we discuss gd T-cell-based therapeutic strategies with a focus on recent developments of bispecific gd T-cell engagers (bsTCEs) and chimeric antigen receptor (CAR) gd T-cells, and point towards approaches that may develop into therapies in the near future ( Figure 1B).

PAST CLINICAL STUDIES WITH Vg9Vd2 T-CELLS
In the year 2000, ABP drugs, already approved to treat patients with excessive bone resorption, were shown to cause systemic Vg9Vd2 T-cell stimulation and to increase their antitumor activity in a preclinical study (26). Following this observation, studies explored ABP treatment as a systemic gd T-cell stimulant or as an ex vivo tool to expand them for subsequent adoptive cell transfer (ACT) for cancer immunotherapy.

PRESENT AND FUTURE STUDIES INVOLVING gd T-CELLS
gd T-Cell-Based Cellular Strategies Allogeneic gd T-Cell Transfer As mentioned above, most gd T-cells recognize target cells independently of HLA antigen presentation, suggesting that allogeneic donor derived gd T-cells can be relatively safe for ACT due to low risk of graft-versus-host disease (GvHD). Taking advantage of this, current strategies exploring the use of ex vivo expanded gd T-cell infusion have shifted towards allogeneic origin ( Table 1). Increased frequency of gd T-cells in leukemia patients that underwent ab-depleted allogeneic stem cell transplantation from partially HLA-mismatched donors, was associated with a higher 5-year and overall survival (OS) (55,56). A single infusion of allogeneic Vg9Vd2 T-cells, expanded ex vivo with ZOL plus IL-2, is being administered in a clinical trial (NCT03533816) to maximize antitumor response and reduce GvHD, after allogeneic hematopoietic cell transplant (alloHCT) and cyclophosphamide for hematologic malignancies. Moreover, allogeneic Vg9Vd2 T-cell infusion after lymphodepletion is being tested independently of alloHCT for hematologic malignancies and solid tumors. Some of these studies have already been completed with no major adverse effects reported, highlighting the safety of Vg9Vd2 T-cell transfer (57,58). Importantly, patients receiving Vg9Vd2 T-cell infusion had increased OS compared to control patients and repeated Vg9Vd2 T-cell infusions resulted in higher OS when compared to single infusion. Future approaches are based on allogeneic gd T-cells derived from healthy donors, either unmodified or CARtransfected (see below) ( Table 2).
Application of non-Vg9Vd2 T-cell subsets, like Vd1 T-cells, is of interest but lagged behind because of lack of proper expansion protocols. In 2016, Almeida et al. described a 3 week culture protocol based on stimulation of gd T-cells from healthy donors or CLL patients with a combination of cytokines and anti-CD3 monoclonal antibody (mAb) clone OKT-3, resulting in 2000fold expansion and 60-80% enrichment of Vd1 T-cells (59). Expanded cells expressed the NK receptors NKp30 and NKp40, displayed cytotoxic activity, produced IFNg, TNFa and no IL-17. Application of this protocol led to the development of different "delta one T" (DOT) cell products. Gamma Delta Therapeutics initiated a first-in-human phase I clinical trial in AML patients after lymphodepletion with fludarabine and cyclophosphamide (NCT05001451) ( Table 1). This study will analyse safety and

Chimeric Antigen Receptor gd T-Cells
Another   and MM (65)(66)(67)(68)(69). The remarkable success of CAR-T-cell therapy revolutionized the field of adoptive cell therapy for treating hematologic malignancies and resulted in numerous ongoing clinical trials. However, CAR-T-cell therapy can be complicated by severe, potentially life-threatening, toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and other 'on-target offtumor' toxicities (70). Moreover, in contrast to the results seen in hematologic malignancies, only limited antitumor effects have been obtained in patients with solid tumors. It was hypothesized that the efficacy of CAR-T-cells could be improved and its side effects mitigated by harnessing the innate properties of gd T-cells as a backbone for CAR. CAR-modified gd T-cells were first described by Rischer et al. (71), demonstrating specific in vitro tumor cell lysis using ZOL-expanded Vg9Vd2 Tcells with CD19-or GD2-directed CARs, followed by other studies confirming these findings using gd T-cells containing CARs against a variety of targets (72)(73)(74)(75)(76)(77). Interestingly, CARmodified Vg9Vd2 T-cells maintained their ability to crosspresent tumor antigens to ab T-cells in vitro, which may prolong the anti-tumor efficacy (76). Furthermore, gd T-cells bearing a CD19-CAR, unlike standard CD19-ab CAR-T-cells, had reactivity against CD19-positive and negative tumor cells in vitro and in vivo, an effect that was enhanced by ZOL (78), suggesting that CD19-directed gd CAR-T-cells may target leukemic cells also after antigen loss and retain pAg specificity via their TCR. More recently, Wallet et al. described the generation of induced pluripotent stem cell-derived gd CAR-Tcells (gd CAR-iT) (79). They demonstrated sustained in vitro tumor cell killing by gd CAR-iT-cells in the presence of IL-15, with markedly less IFN-g and other inflammatory cytokines being produced compared to conventional ab CAR-T-cells, potentially resulting in lower risk of CRS. Moreover, a single dose of gd CAR-iT-cells resulted in potent tumor growth inhibition in a xenograft mouse model (79). Table 2 summarizes the companies currently developing gd CAR-T-cells.
Pre-clinical research on gd CAR-T-cell based therapy initially focused on Vg9Vd2 T-cells, due to their dominant frequency in blood and their unique pAg response that allowed the specific expansion of this subset (80). Makkouk et al. recently showed the first example of genetically modified Vd1 T-cells. They expanded PBMC-derived Vd1 T-cells using an agonistic anti-Vd1 antibody and genetically modified them to express a GPC-3 targeted CAR and to secrete IL-15 (81). In a HepG2 mouse model, these allogeneic Vd1 CAR-T-cells primarily accumulated in the tumor and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. ADI-001 consists of CD20-targeting Vd1 CAR-T-cells generated by a similar procedure by Adicet Bio (82) and is currently being used in a phase I clinical trial (NCT04735471). Recently reported interim data from this dose-escalation study showed complete responses in two and a partial response in one out of four evaluable patients already with low doses (30x10 6 cells) of ADI-001, indicating that relatively low amounts of gd T-cells may suffice for activity (press release). To date, no dose-limiting toxicities, GvHD, or grade 3 or higher CRS has been reported. These encouraging first results underscore the potential of Vd1 CAR-T-cell therapy in the clinic. A complete overview of the ongoing clinical trials evaluating CAR-modified gd T-cells is listed in Table 1.

Antibody-Based Strategies
Imcheck develops ICT01, a Vg9Vd2 T-cell activating humanized IgG1 with a silent Fc that binds to all three BTN3A isoforms to trigger Vg9Vd2 T-cell activation and increased cytotoxicity against BTN3A + tumor cell lines from diverse origin (21). However, this approach is not tumor specific as BTN3A is broadly expressed and could also be hampered by soluble BTN3A molecules potentially acting as decoy receptors (83). In immunodeficient NSG mice, treatment with ICT01 resulted in in vivo activation of adoptively transferred human Vg9Vd2 T-cells and delayed outgrowth of the AML cell line MOLM14 (84). The EVICTION trial is a Phase I/IIa clinical trial currently testing the effect of ICT01 in relapsed/ refractory advanced-stage hematologic malignancies as a monotherapy and in a broad range of solid tumors as monotherapy or in combination with pembrolizumab (NCT04243499). Preliminary results show a good safety profile with activation of Vg9Vd2 T-cells and increased tumor infiltration in one melanoma patient. Stable disease has been achieved in 31% of patients treated with ICT01 as a monotherapy and in 62% in combination with pembrolizumab (84).
BsTCEs have emerged as a promising therapeutic approach for immune-oncology (85) and consist of a tumor antigen binding antibody linked to a T-cell engaging antibody fragment aiming to crosslink tumor cells and T-cells to elicit T-cell-mediated anti-tumor cytotoxicity (86,87). Most efforts to generate bsTCEs have made use of CD3 as a T-cell engaging domain due to its role in T-cell activation. For CD3-based TCEs, proteins that are uniquely expressed or specifically overexpressed by tumor cells are the most attractive candidates for targeting, as this reduces on-target off-tumor toxicity. After approval of the CD19-CD3 bsTCE blinatumomab (88), multiple CD3-directed TCEs have been developed (89), but in many cases development has been complicated by the occurrence of adverse events such as on-target off-tumor toxicity, CRS or ICANS, highlighting the need for more tumor-selective targeting (90)(91)(92). Considering the clinical safety observed following systemic gd T-cell activation and gd T ACT, specific engagement of gd T-cells using gd bsTCEs might have an improved safety profile due to their tumor selectivity compared to CD3-bsTCEs. By avoiding detrimental co-activation of regulatory CD3 + T-cells observed with CD3 pan T-cell engagers (93) and their ability to bridge and engage components of both the innate and adaptive immune system, gd bsTCEs could potentially result in increased antitumor activity.
Several gd T-cell engaging formats are being developed and evaluated preclinically. Vg9-TCR specific engagers directed against Her2 (94)(95)(96) and CD123 (97) were shown to cause killing of Her2 expressing cell lines and AML cell lines, respectively. The GADLEN platform (Shattuck Labs) consists of fusion proteins containing BTN heterodimers, to engage and activate Vg9Vd2 T-cells, bound to a tumor targeting scFv domain through an Fc linker (98). Vd1 bsTCEs are also being developed by Adaptate Biotherapeutics. Heavy chain only antibodies occur naturally in camelids (99). Their antigenbinding fragments or variable heavy chain-only antibodies ( V H H ) , a r e s m a l l , s t a b l e a n d wi t h l o w i n h e r e n t immunogenicity (100,101). Lava Therapeutics`Gammabody ™ platform combines Vd2-specific and tumor-targeting VHHs as modules to generate bsTCE (102)(103)(104)(105). In pre-clinical studies, Gammabody ™ molecules targeting CD40, CD1d and EGFR efficiently engage Vg9Vd2 T-cells to kill tumor cells expressing these antigens (102)(103)(104)(105). Two Gammabody ™ molecules, are currently evaluated in clinical trials. LAVA-051, a Gammabody ™ targeting CD1d is tested in a Phase I/IIa clinical trial (NCT04887259) in patients with therapyrefractory CLL, AML or MM. Preliminary data of the first 3 cohorts from this study showed a thus far good safety profile with no dose-limiting toxicities or CRS. In addition, LAVA-1207, a Gammabody ™ targeting PSMA is tested in a phase I/IIa clinical trial (NCT05369000) in patients suffering from therapyrefractory metastatic castration-resistant prostate cancer. Table 2 summarizes companies developing antibody-based gd T-cell therapies, and Table 1 contains clinical trials involving antibody-based gd T-cell approaches.

Alternative gd T-Cell-Related Strategies
A new gd T-cell based approach being tested in clinical trials is DeltEx drug-resistant immunotherapy (DRI). IN8Bio`s first DeltEx DRI product, INB-200, consists of expanded autologous Vg9Vd2 T-cells genetically modified to express a methylguanine DNA methyltransferase (MGMT). MGMT confers them resistance to temozolomide (TMZ) allowing for simultaneous treatment with TMZ and immunotherapy (106). TMZ, which is the current standard of care for glioblastoma multiforme (GBM) together with radiotherapy after resection, might sensitize tumor cells to gd T-cell recognition through upregulation of NKG2D ligands but it also causes lymphocytopenia that is avoided by MGMT expression (107). An ongoing clinical trial (NCT04165941) is testing intracranial administration of INB-200 to the tumor site after surgical resection, followed by TMZ treatment ( Table 1). All 4 GBM patients enrolled in this study have been reported to exceed the expected PFS for TMZ alone treatment. This technology is based on expansion and modification of autologous gd T-cells, however, other DeltEx DRI based on allogeneic gd T-cells (INB-400) and gd CAR-Tcells (INB-300) are being developed.
Interestingly, although Vd1 + T-cells have cytotoxic capacity, Vd1 + TIL associate with poor prognosis in certain malignancies, possibly through production of IL-17 (6,32). LYT-210 is a mAb directed towards the Vd1 + TCR with the aim of eliminating these pathogenic cells ( Table 2). Gamma-delta TCR bispecific molecules (GABs) combine the extracellular domain of the Vg9Vd2 TCR fused with a CD3 binding domain, allowing conventional T-cells to recognize the presence of pAg on tumor cells (108). In the presence of GABs, ab T-cells recognized and killed the squamous cell carcinoma cell line SCC9 in a pAg dependent manner and produced increased amounts of IFNg when exposed to patient-derived AML blasts but not with healthy hematopoietic cells indicating preferential recognition of tumor cells.
Two phase I dose-escalation clinical trials (NCT04688853; NTR6541) initiated by Gadeta are assessing the safety and tolerability of ab T-cells engineered to express a defined Vg9Vd2 TCR (TEGs) in relapsed/refractory AML, MM, and high-risk myelodysplastic syndrome patients. These T-cells combine the tumor specificity of gd T-cells with the tumor cell killing potential of ab T-cells and show promising antitumor reactivity both in vitro and in vivo. Furthermore, chimeric PD-1 receptor (chPD1) gd T-cells, turn PD-1 immune suppression into T-cell activation (109). The chPD1 gd T-cells selectively killed PD-L1 + tumor cells in a xenograft murine model, without lysis of normal PD-L1 + cells or significant elevation of CRS-related cytokines. The authors reported that chPD1 gd T-cell therapy will be assessed in a phase I/II clinical trial.

CONCLUSION
Past clinical trials have demonstrated that systemic activation of Vg9Vd2 T-cells or adoptive transfer of autologous Vg9Vd2 T-cells were well tolerated and could trigger antitumor immunity. These studies have been followed by a number of trials based on Vg9Vd2 and the first study with Vd1 allogeneic T-cell transfer, which would allow for donor-derived therapies. Up to this date, these trials have not resulted in major adverse effects. Most strategies that are currently under evaluation profit from the safety of gd T-cell activation and incorporate tumor-targeting mechanisms, e.g. CARs or bsTCEs, which might be key to obtain more robust and consistent clinical responses. Initial results from these targeted approaches, both cell and antibody-based, show great promise and confirm the safety of Vg9Vd2 and Vd1 T-cell-based strategies. However, cell-based products present challenges that are not shared by antibody-based therapies, such as high cost, difficulty of production or need of specialized facilities, and preparatory lymphodepleting chemotherapy regimens. In the near future, the results obtained by the trials described in this review will determine whether the potential of gd T-cells can be translated into clinical benefit.

AUTHOR CONTRIBUTIONS
JS-E and MJ wrote the manuscript. HV co-wrote and reviewed the manuscript. LK, PP, EE, BW and TG reviewed the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
The authors declare that this study received funding from LAVA therapeutics. The funder had the following involvement with the study: providing research funding to Amsterdam UMC and in designing, writing and revising the text of the mini-review.